PET PROTEIN: Methods of Purification

This assignment will require extensive literature searching to locate prime sources in books and journals.

The protein of industrial/medical importance which you would be using in this assignment is Hirudin.

You will need to select a specific species variant that is used in a medical or industrial application.

Methods of Purification (Written report due MONDAY Apr7 11am Sydney time)
Find out about:
1. The primary structure (amino acid sequence).

• Prepare your own figure (not a journal extract) of the protein sequence, clearly showing the numbering relevant to the mature sequence. Outline and label any sub-regions or domain units important in this protein. Indicate on this figure the disulfide connections (if any), as well as any amino acids known to be glycosylated.

 2. The physico-chemical properties of pI, MW, GRAVY value of the protein that are important considerations for a purification protocol.

• Use the tools in ExPASy to analyse your sequence. Check you have the mature sequence of your protein (i.e. remove any signal peptide)
• If a glycoprotein, detail the full chemical nature of the carbohydrate portion.
• Present a hydropathy plot of the marure seqeunce of your protein (see ProtScale: web.expasy.org/protscale/).
• Discuss (i) the features of this plot with relation to the specific amino acid sequence and (ii) how it can be used to determine the GRAVY (grand average hydropathy) value.
• Comment on the link between the pI, GRAVY value and amino acid composition.

3. How the protein was initially purified from its natural source when it was first discovered.

• Search the literature to source the original discovery route for this protein.
• Present a summary for this first purification as a simplified flow chart. It must present a summary overview of the chemical partitioning events, not detailed methods & reagents. Clearly label discarded and retained fractions at each partition.
• Within your report, give a detailed description of the molecular events and chemistry of each separation step in the specific procedure, at the same detailed level as theory from lectures.
• Where possible, include and discuss column traces or stained gel images that indicate the purification achieved at specific steps. Note: your flowchart must include branch points to clearly show how the mixture is successively partitioned to remove unwanted components and achieve purification.

4. How is the same protein now prepared for an industrial use, or in recombinant form?

• For what purpose is your protein produced by industry?
• Present a detailed protocol for its purification as a flow chart, as above.
• Provide a detailed description of the molecular events and chemistry of each step in the procedure.
• Where possible, include and discuss column traces or stained gel images that indicate the purification achieved at specific steps.
Note: your flowchart must include branch points to clearly show how the mixture is successively partitioned to remove unwanted components and achieve purification. 5. Finish your written submission with a bibliography citing all primary sources, correctly formatted.

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